ATP6L增强结肠癌细胞干细胞样特性的相关机制及其临床意义

ATP6L, c subunit of proton pump V-ATPase V0 domain, plays vital role in H+ transmembrane transportation and is involved in maintaining the abnormal acidic microenvironment of tumor. It has been reported that ATP6L could promote tumor growth and metastasis. However, its role in regulating stemness of tumor cells remains to be elucidated. In our previous study, we found that ATP6L protein overexpressed in the poor-differentiated colon cancer tissues and was closely related to tumor metastasis and recurrence. Furthermore, overexpression of ATP6L enhanced tumor-initiating capability of colon cancer cells，indicating that ATP6L may enhance stem cell-like characteristics of cancer cells. In this proposal, we will study the effect of ATP6L on regulating stemness of colon cancer cells at cellular level, animal model level and clinical case analysis, respectively. Meanwhile, we found that ATP6L was closely related to activation of Wnt/β-catenin signaling,.which is one of the most important signaling pathways in regulating stemness of tumor cells. We will further investigate the possible molecular mechanism of how ATP6L effect on Wnt/β-catenin signaling activity and explore that whether ATP6L enhances stemness of tumor cells by activating Wnt/β-catenin signaling. In summary, the in-depth study of ATP6L protein and its mechanism, would help us.understanding the important role of acidic microenvironment during tumor initiation and development, and provide novel ideas for tumor pathogenesis, as well as potential target for the treatment of colon cancer.

ATP6L是质子泵V-ATPaseV0功能域的c亚单位，在细胞进行H+ 跨膜转运过程中至关重要，参与维持肿瘤异常酸性微环境。ATP6L在肿瘤细胞中的异常表达促进肿瘤的生长、转移，但其对肿瘤细胞干细胞样特性的作用至今尚未有报道。我们的前期研究发现ATP6L在分化差的结肠癌组织中高表达并与肿瘤转移、复发密切相关，且ATP6L高表达的结肠癌细胞成瘤能力显著增强，提示ATP6L可能增强肿瘤细胞干细胞样特性。本课题拟在细胞水平、动物模型和临床病例分析三方面系统研究ATP6L对结肠癌细胞干细胞样特性的调控作用。另外，我们发现ATP6L与Wnt/β-catenin信号通路活化密切相关，后者是调控肿瘤细胞干细胞样特性的重要信号通路。我们将进一步研究ATP6L调控Wnt/β-catenin信号通路活化的分子机制，并探讨ATP6L是否通过促进Wnt/β-catenin信号通路活化而增强肿瘤细胞干细胞样特性。

本项目主要探讨肿瘤酸性微环境调节蛋白ATP6L 在人结肠癌组织中表达的临床病理意义及预后价值；明确其促进结肠癌细胞 Wnt/β-catenin 信号通路的活化及增强结肠癌细胞“干样”特性的作用，寻找酸性微环境对肿瘤细胞“干样”特性的关键调控因子和相关分子机制。主要研究结论如下：（1）ATP6L在分化差的结肠癌组织中表达较高，ATP6L高表达的患者肿瘤转移、复发率更高且提示预后不良；（2）ATP6L过表达结肠癌细胞Lgr5表达增加、体外细胞成球及耐药实验、动物水平肿瘤成瘤实验表明 ATP6L 能够增强结肠癌细胞“干样”特性。（3）ATP6L过表达结肠癌细胞β-catenin 存在向核内集中表达的趋势，且 c-myc、cyclinD1等蛋白表达上调，提示ATP6L促进结肠癌细胞 Wnt/β-catenin 信号通路活化；（4）通过Dkk1抑制Wnt/β-catenin 信号通路可逆转 ATP6L 对结肠癌细胞“干样”特性的增强作用，提示Wnt/β-catenin 信号通路在增强结肠癌细胞干细胞样特性方面具有重要作用。此外，干细胞样肿瘤细胞对于组织中非宿主血管内皮来源的肿瘤血管新生具有重要作用。血管生成拟态（VM）作为一种不依赖内皮细胞，而是由肿瘤细胞构成的血供模式。参与构成VM结构的肿瘤细胞具有胚胎样可塑性、高度侵袭和转移能力等干细胞样特征，可通过上皮间充质转化（EMT）等转分化过程直接参与上皮来源性肿瘤的血管新生，是肿瘤细胞干样特性的具体体现，也是促进肿瘤恶性演进的重要事件。因此，课题组对研究内容进行了扩展，进一步探讨了ATP6L表达与结肠癌组织中不同血管生成模式间的关系，Wnt/β-catenin 信号通路调节蛋白Wnt3a及Dkk1对结肠癌细胞EMT及VM形成的作用。研究发现，ATP6L表达与结肠癌组织中VM密切相关，能够促进分化较差的结肠癌HCT116细胞在体外形成管道样结构并使VE-cadherin表达增强，提示ATP6L可能增强结肠癌细胞的转分化能力而促进VM形成，促进结肠癌恶性演进；Wnt3a促进结肠癌细胞EMT并促进结肠癌细胞向血管内皮方向转分化，促进VM形成，而Dkk1可抑制结肠癌VM形成，提示Wnt/β-catenin 信号通路在增强结肠癌细胞干细胞样特性、促进结肠癌细胞EMT及肿瘤组织中VM形成具有重要作用。